双重PCR快速检测KPC和NDM耐药基因

Duplex PCR for rapid detection of the drug-resistant genes KPC and NDM

目的检测耐药基因的核酸技术,已成为有效监测和控制耐药菌扩散的强有力工具之一。建立一种快速检测KPC和NDM两种耐药基因的方法,以指导临床用药和监控院内细菌耐药性流行情况。方法根据肺炎克雷伯菌碳青霉烯酶(KPC)和新德里金属β-内酰胺酶(NDM)的基因保守区,设计PCR反应的引物,优化扩增体系,建立同时检测耐药基因KPC和NDM的方法,分析方法的灵敏度和特异性,并应用于铜绿假单胞菌和肺炎克雷伯菌株的检测。结果 KPC和NDM基因的序列比对结果与原序列相比一致性为100%。KPC-2阳性标准品和标准菌株肺炎克雷伯菌ATCC BAA-1705均出现预期大小一致的目的片段(151 bp); NDM-2阳性标准品与NDM阳性肺炎克雷伯菌出现与预期大小一致的目的片段(261 bp),均无非特异性扩增。经对PCR产物进行测序验证,产物序列与耐药基因KPC-2和NDM-1序列一致性为100%。双重PCR检测耐药基因KPC和NDM的最低可检测浓度分别为7×102和5×102copies/反应。13株对碳青霉烯耐药的肺炎克雷伯菌株中12株检测到耐药基因,检出率为92.3%,其中KPC阳性10株(83.3%),NDM阳性2株(16.7%),其余10株碳青霉烯敏感的肺炎克雷伯菌株未检测到KPC和NDM; 13株铜绿假单胞菌中6株对碳青霉烯耐药的菌株中2株检测到耐药基因,均为KPC,检出率为33.3%,其余7株均未检测到KPC和NDM。结论双重PCR方法能够快速有效地用于KPC和NDM两种耐药基因的检测,并具有灵敏度高、特异性强等优点,对于指导临床用药与监控院内细菌碳青霉烯类抗生素耐药性的传播具有重要意义。

Objective The nucleic acid technology for detecting drug-resistant genes has become one of the powerful tools for monitoring and controlling the spreading of drug-resistant bacteria. This study was to establish a method for rapid detection of the drug-resistant genes KPC and NDM and provide some guidance in clinical drug use and monitoring the prevalence of drug-resistant bacteria in the hospital. Methods According to the conserved regions of Klebsiella pneumoniae carbapenemase （KPC） and New Delhi metallo-β-lactamase （NDM）, we designed the primers of duplex PCR, optimized the amplification system and established a method for simultaneous detection of the drug-resistant genes KPC and NDM . Then, we analyzed the sensitivity and specificity of the method and applied it to the detection of Pseudomonas aeruginosa and Klebsiella pneumoniae. Results The sequences of KPC and NDM exhibited a 100% consistency with those of the original ones. Target fragments of the desired size of 151 bp were detected in the KPC-2 positive standard and Klebsiella pneumoniae ATCC BAA 1705 standard strains, and those of the desired size of 261 bp were observed in the NDM-2 positive standard strain and NDM-positive pneumococcal bacteria, neither with non-specific amplification. Sequencing of the PCR products showed a 100% consistency between the sequences of the products and those of the drug-resistant genes KPC-2 and NDM-1 . The detectable limits of KPC and NDM for duplex PCR were 7×10^2 and 5×10^2 copies per reaction respectively. Drug-resistant genes were detected in 12 （92.3%） of the 13 carbapenems-resistant strains, including 10 KPC-positive （83.3%） and 2 NDM positive ones （16.7%）, but neither KPC nor NDM in the other 10 carbapenems-sensitive strains. In the 13 strains of Pseudomonas aeruginosa, KPC was detected in 2 （33.3%） of the 6 carbapenems-resistant ones, but neither KPC nor NDM in the other 7. Conclusion The duplex PCR method can be used for rapid and effective detection of the drug-resistance genes KPC and NDM , with the advantages of high sensitivity and specificity, and is therefore of great significance for guiding clinical drug use and monitoring the spreading of carbapenems-resistant bacteria in the hospital.